Antibody libraries are useful to identify antibodies of interest and to screen for novel antibody-antigen complexes. For example, the Mehta I and Mehta II single chain antibody libraries contain over 27 billion non-immune human antibodies. Previously, such libraries have been screened using phage display technology. The adaptation of antibody presentation from phage display to mammalian display requires new vector delivery systems.
Vectors that have been employed to deliver exogenous nucleic acids include both DNA viral vectors and RNA viral vectors. For example, DNA vectors include pox vectors such as orthopox or avipox vectors (see, e.g., U.S. Pat. No. 5,656,465), herpes virus vectors, such as herpes simplex I Virus (HSV) vectors (See, Geller, A. I. et al., J. Neurochem. 64:487 (1995); Lim, F., et al., DNA Cloning: Mammalian Systems, D. Glover, Ed., Oxford Univ. Press, Oxford, England (1995); Geller, A. I. et al., Proc. Natl. Acad. Sci., U.S.A. 90:7603 (1993); Adenovirus vectors (Legal Lasalle et al., Sci. 259-988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol., 69:2004 (1995)); and Adeno Associated Virus Vectors (See, Kaplitt, M. G., et al., Nat. Genet. 8; 148 (1994)). Retroviral vectors include vectors obtained from Moloney murine leukemia viruses (MMLV) and human immunodeficiency viruses (HIV) (See, U.S. Pat. No. 5,665,577).
Various vectors have characteristics that make them desirable for certain applications. For example, a retroviral vector can be used to infect a host cell and have the genetic material integrated into that host cell with high efficiency. One example of such a vector is a modified Moloney murine leukemia virus (MMLV), which has had its packaging sequences deleted in order to prevent packaging of the entire retroviral genome. However, that retrovirus does not transduce resting cells. Additionally, since many retroviruses typically enter cells via specific receptors, if the specific receptors are not present on a cell or are not present in large enough numbers, the infection is either not possible or is inefficient. Safety issues have also been raised following outbreaks of wild-type viruses from the recombinant MMLV producing cell lines, i.e., reversions.
A vector derived from an adenovirus can infect a wide range of cells. However, the transferred genetic material is not integrated. Therefore, any expression or screening procedures are limited to the time when the transferred genetic material is episomal. Thus, studies can only occur for a relatively short period of time.
Recently, attention has focused on lentiviral vectors such as those based upon the primate lentiviruses, e.g., human immunodeficiency viruses (HIV) and simian immunodeficiency virus (SIV). By using a pseudotyped vector (i.e., one where an envelope protein from a different species is used), problems encountered with infecting a wide range of cell types can be overcome by selecting a particular envelope protein based upon the type of cell to be infected. Additionally, in view of the complex gene splicing patterns seen in lentiviruses such as HIV, multivalent vectors (i.e., those expressing multiple genes) having a lentiviral core, such as an HIV core, are expected to more efficiently express nucleic acids of interest.
The use of lentiviral vectors, such as those based on primate lentiviruses, (e.g., HIV and SIV), to perform large-scale mammalian transfections offers several advantages over retroviral vectors. Lentiviral vectors containing genes encoding antibodies can infect quiescent cells and proliferating cells both in vivo and in vitro. (See, Reiser et al., PNAS 93:15266-15271 (1996); Naldini et al., Science 272:263-267 (1996)). Additionally, lentiviral vectors allow constitutive or induced expression of heterologous polypeptides, thus providing for the production of antibodies in culture and in animal.
In recent years, attention has been directed to developing large libraries typically consisting of monoclonal antibodies or peptides. For example, antigen binding antibody fragments have been expressed on the surface of filamentous phage (G. P. Smith, Science 228: 1315 (1985)), and used to create large libraries of such antibodies—e.g., 107 members or more, referred to as phage display libraries. In phage display libraries, the carboxyl-terminal end of the Fd or Fv region is tethered to a fragment of a phage coat protein, which anchors, for example, Fab fragment to the surface of the phage. Antibody display systems have been described in non-mammalian cells, such as E. coli (Daugherty et al, Protein Eng 1999, 12:613-621), yeast (Shusta et al. Nat Biotechnol 2000, 18:754-759), and baculoviral infection of Sf9 insect cells. Despite the advantages of being able to express antibodies on the surface of phage or non-mammalian cells, it was recognized that the expressed antibodies can often fail to fully retain their binding activity (Choe et al. Cell 2003, 114; 161-170; Huang et al. PNAS 2004, 101:2706-2711).